L’identification des bases moléculaires de la tolérance au gel présente une grande importance tant sur le plan fondamental qu’appliqué. Medicago truncatula est une légumineuse modèle pour les espèces tempérées. Un QTL majeur de tolérance au gel après acclimatation au froid (Mt-FTQTL6), expliquant 40% de la variation phénotypique, a été détecté sur le chromosome 6 de cette espèce. En parallèle, un QTL pour le même caractère (WFD 6.1/FD164.c) a été identifié sur le groupe de liaison équivalent chez Pisum sativum. Cette thèse a pour objectifs de confirmer la synténie des régions chromosomiques contenant Mt-FTQTL6 et WFD 6.1/FD164.c et d’identifier des gènes candidats positionnels pour Mt-FTQTL6. Les premiers efforts ont permis de localiser Mt-FTQTL6 dans un intervalle de 3,7 cM qui coïncide avec une zone du génome de Medicago truncatula dont l’assemblage reste incomplet. Les ressources génomiques de Glycine max ont été ensuite exploitées. Cinq marqueurs géniques ont permis d'ancrer les régions chromosomiques de Mt-FTQTL6 et WFD 6.1/FD164.c. Des clones BAC correspondant à 15 marqueurs (sondes) ont été assemblés en 6 contigs couvrant l’intervalle de confiance de Mt-FTQTL6. Des lignées F7 ou F8, recombinantes au niveau de cet intervalle, ont été identifiées et phénotypées pour la tolérance au gel en conditions contrôlées. L'intervalle de confiance de Mt-FTQTL6 a ainsi été réduit à une région de 0,4 cM contenant 20 gènes parmi lesquels 12 gènes CBF/DREB1 en tandem. La variation allélique pour 11 gènes CBF/DREB1 a été mise en évidence chez les parents de la population de cartographie.La validation fonctionnelle est maintenant envisageable chez Medicago truncatula et Pisum sativum. / Unraveling the molecular bases of freezing tolerance is of great importance both at the fundamental and applied levels. Medicago truncatula is a model legume for studies concerning cool-season species. A major freezing tolerance QTL after cold acclimation (Mt-FTQTL6), accounting for 40% of the phenotypic variation, has been identified on chromosome 6 of this species. Interestingly, a QTL for the same trait has been mapped on the corresponding linkage group in Pisum sativum (WFD 6.1/FD164.c). The present thesis aimed to confirm synteny between Mt-FTQTL6 and WFD 6.1/FD164.c harboring regions and to subsequently identify positional candidate genes for Mt-FTQTL6. Using BAC-derived markers, Mt-FTQTL6 has been first located in a 3.7-cM interval, coinciding with an assembly physical gap. Mt-FTQTL6 co-orthologous blocks in Glycine max were identified and exploited to develop additional markers. Five common gene-based markers were obtained between Mt-FTQTL6 and WFD 6.1/FD164.c chromosomal regions. Positive BAC clones for 15 different markers (probes) were assembled in 6 BAC contigs linked to Mt-FTQTL6. Homozygous F7 or F8 recombinant lines at Mt-FTQTL6 were identified and evaluated for freezing tolerance under controlled conditions. The QTL confidence interval was subsequently delimited to a 0.4 cM-region that contains 20 protein-coding genes including 12 tandemly-arrayed CBF/DREB1 genes. Isolation of 11 out of the 12 CBF/DREB1 genes from both parents of the mapping population was successfully achieved. Efforts will be next needed for functional validation in Medicago truncatula and Pisum sativum.

Breast cancers frequently metastasise to the skeleton where they cause osteolytic bone destruction. Effective treatment of bone metastasis remains a considerable clinical challenge. In the UK around 70% of the 12,000 patients that die from breast cancer annually have bone metastasis. Whilst existing therapies provide some pain relief, by limiting the tumour-mediated bone degradation, bone metastases are presently incurable. There is therefore an urgent need to develop therapies to prevent bone metastatic breast cancer. The transcription factor complex Runx2/CBFβ is a key regulator of bone development and is aberrantly expressed in breast cancer, leading to up-regulation of bone metastasis-associated genes. Previous work has demonstrated that Runx2/CBFβ determines the invasive phenotype of metastatic breast cancer cells and is required for the expression of metastatic genes. The Runx2/CBFβ complex also has a role in normal breast gene expression, activating expression of the milk protein β-casein in response to hormones. However, little is known about the normal role of Runx2/CBFβ in breast cells. The overarching aim of this project was to determine the role of Runx2/CBFβ in metastasis and identify the target genes that determine the metastatic phenotype. In order to understand the role of Runx2/CBFβ in breast cancer, initial experiments were performed to determine the role of Runx2/CBFβ in normal breast cells. A 3D culture system was established to examine the role of Runx2/CBFβ in regulating gene expression in non-cancerous differentiated epithelial breast cells. Attempts were also made to determine the Runx2/CBFβ target genes after lactogenic hormone stimulation. Unfortunately siRNA knockdown of Runx2 was incompatible with hormonal stimulation. However, 3D cell culture of normal mammary gland cell line HC11 showed Runx2 was expressed throughout the development of mammary acini structures. In addition the expression of CBFβ was confirmed in these cells. Having established the 3D culture system, experiments were subsequently performed to examine the role of CBFβ in the metastatic breast cancer cell line MDA-MB-231. These experiments demonstrated that depletion of CBF has a remarkable effect on the phenotype of the cells, leading to the development of mammary acini structures normally formed by non-cancerous breast cancer cell lines. Thus, depletion of CBF results in a reversion to an epithelial phenotype, suggesting that CBF is required to maintain the epithelial to mesenchymal transition (EMT). RT-PCR analysis also revealed changes in the expression of EMT marker genes. We also demonstrated that the EMT reversion could be rescued by re-expressing an inducible form of CBFβ. These data suggest that CBFβ is required to maintain the mesenchymal phenotype of metastatic breast cancer cells. Finally, a microarray analysis of MDA-MB-231 cells was performed to identify Runx2/CBFβ target genes that might contribute to the mesenchymal phenotype. Cells depleted of CBFβ and grown in 3D revealed reduced expression of IL11. This is known to be involved in bone remodelling. Inspection of the IL11 promoter revealed potential DNA binding sites which confirmed binding to Runx2 using EMSA.

No description available.

Wheat (Triticum aestivum L.) is occasionally exposed to low temperature during flowering and huge economic losses can occur especially in some key production countries such as Australia. Although it is generally predicted that there will be a rise in global temperature there are still predicted to be risks associated with low temperature for temperate crops. Post head emergence frost damage remains a major constraint to increasing wheat production. Five Iraqi varieties (Abu-Ghariab, Fatah, Sham6, IPA95 and IPA99) were screened for their frost hardiness (LT50). Abu-Ghariab was chosen for further investigations along with the European cv. Claire because the acclimated frost hardiness level in these two varieties (LT50 -8.07 and -8.01°C for Claire and Abu-Ghariab respectively) was found to be significantly lower than the other varieties in this study. Several techniques were employed including REC%, IR thermography and molecular analysis of cold acclimation in order to characterise the frost resistance of those two varieties. REC% revealed that both Claire and Abu-Ghariab could tolerate some freezing when the spikes were just visible (ZCK 51-60) with an acclimation shift of the LT50 of -1.6 and -2.11°C respectively but this was not apparent at later growth stages. Based on molecular analysis, cold acclimation was shown to be activated at ZCK 51-60 in both varieties. Cbf14 was expressed after 8 hours exposure to acclimatising temperatures (4°C) and then declined to a low, but still up-regulated level in both varieties and this led to expression of the COR15a protein. These molecular changes correlated with the frost tolerance recorded at ZCK 51-60. It was concluded that the possibility existed to up-regulate cold acclimation after spike emergence if there was enough environmental stimulus. Molybdenum (Mo) was demonstrated to work synergistically with low temperature in increasing the expression of Cbf14 and COR15a. The European wheat cv. Claire showed a higher capacity (-8.14°C) to be acclimated than Iraqi wheat (-7.40°C) under the effect of both Mo and acclimation temperatures. Mo alone increased the expression of Cbf14 in both varieties but did not increase the frost tolerance. Observations of ice nucleation using an infrared thermography (IR) revealed that supercooling is highly likely in spikes and some spikes avoided frost damage even when the temperature fell to -12°C. It was observed that the proportion of frozen spikes was 22.8% while the remainder supercooled. Spraying plants with distilled water was not effective in facilitating ice nucleation in wheat spikes. Observations also revealed that spikes that did freeze started freezing at temperatures of -4 to -5 °C close to temperature of the putative constitutive frost hardiness of un-acclimated wheat and it is suggested that this may reflect that many laboratory freezing experiments may not actually freeze until -5°C. The anthesis stage of wheat was found to be the most vulnerable stage and it needs to be given more attention in terms of research to up-regulate cold acclimation. Frost damage to wheat during flowering continues to be a serious problem in certain production areas and therefore continued effort in characterising and finding suitable solutions to this are imperative.

A comprehensive review of the nasal route of administration, in particular the nasal drug delivery system has been presented. The physicochemical properties, mode of action and pharmacology of H1-receptor antagonists, in particular cyclizine HCl, have been highlighted. The techniques for the assessment of toxicity (in-vitro ciliary beat frequency (CBF) studies for human nasal explants and morphology studies of the rat nasal mucosa), synthesis of cyclizine lactate, solubility studies of both cyclizine HCI and cyclizine lactate, viscosity determination of the gel formulated and assessment of the deposition and distribution of the hydroxypropylmethyl cellulose (HPMC) dispersions within the human nasal cavity model were conducted. In this study, preliminary studies on the toxicity of the various formulation components (excipients and active ingredient) were carried out. Results from these studies indicated that for both the excipients and the drug, pH significantly affects the ciliary motility hence all ciliary beat frequency determinations were conducted at nasal pH. Furthermore, effects of the various concentrations (0.0625%(w/v), 0.125%(w/v), 0.25%(w/v), 0.5%(w/v) and l%(w/v)) of the excipients on ciliary motility were investigated. Transmission electron microscopy (TEM) studies proved useful in evaluating the integrity and changes in the surface morphology of the rat nasal mucosa post treatment with the various excipients (carboxymethyl cellulose, hydroxypropylmethyl cellulose, trimethyl chitosan 36.3% DQ, Carbopol P934 and polysorbate-80) at varying concentrations. Of the excipients investigated, hydroxypropylmethyl cellulose (HPMC) showed ciliofriendliness since there was no apparent ultra structural damage, although a slight decrease in ciliary beat frequency (CBF) was observed at the highest viscosity. Moreover, hydroxypropylmethyl cellulose (HPMC) is said to be a bioadhesive excipient, which would therefore confer its bioadhesive properties to the intranasal preparation to enhance the retention time between the absorbing mucosa and the drug and hence increase nasal drug absorption. This excipient was therefore selected as the ideal for use in the formulation of the intranasal preparation. The aqueous solubility of a drug plays an important role in nasal administration since it is required that the drug component be applied in a limited volume of about 200pl. To enhance the aqueous solubility of the sparingly water-soluble cyclizine HCl, a lactate salt was synthesised and characterised. This compound was found to be highly soluble in water. The intranasal preparation was therefore manufactured using the lactate form of cyclizine. A single blind study was conducted to determine and compare the pharmacokinetic parameters for both Valoid oral tablets containing 100mg cyclizine HCl (reference drug) and cyclizine lactate intranasal preparation 125mglml (study drug). The results obtained indicated a significant improvement in the bioavailability of cyclizine. For oral administration Cmax = 200.79ng/ml at tmax = 5.57h and for the intranasal preparation Cmax = 5354.22ng/ml at tmax = 1.59h. A 19.2-fold increase in drug bioavailability was observed after intranasal administration (AUCin = 122860.70ng/ml/h) compared with oral administration (AUCpo = 5943.48ng/ml/h). This enhanced bioavailability through nasal administration indicated that enhanced nasal drug absorption and hence increased bioavailability not only depends on the favourable anatomical and physiological characteristics of the nasal mucosa but possibly on the inherent physico-chemical characteristics of the drug molecule and the formulation components. Thus chemical modification of the sparingly water-soluble cyclizine HCl to the highly water-soluble cyclizine lactate facilitated the dissolution of more solute in a limited volume of solvent. This new feature therefore may have impacted positively to the transport of cyclizine across the nasal mucosa. Furthermore, the hydroxypropylmethyl cellulose (HPMC), component of the formulation, could have conferred its mucoadhesive properties to the preparation. Perhaps it increased the retention time of the dosage form within the nasal passages through bond formation with the nasal mucosa thereby increasing the contact time between the absorbing mucosa and the dosage form. This interaction between the mucoadhesive and the nasal mucosa may have resulted in the modification of tissue permeability (possibly transient opening of the tight junctions) and eventual increase in the drug penetration/absorption. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2004.

A comprehensive review of the nasal route of administration, in particular the nasal drug delivery system has been presented. The physicochemical properties, mode of action and pharmacology of H1-receptor antagonists, in particular cyclizine HCl, have been highlighted. The techniques for the assessment of toxicity (in-vitro ciliary beat frequency (CBF) studies for human nasal explants and morphology studies of the rat nasal mucosa), synthesis of cyclizine lactate, solubility studies of both cyclizine HCI and cyclizine lactate, viscosity determination of the gel formulated and assessment of the deposition and distribution of the hydroxypropylmethyl cellulose (HPMC) dispersions within the human nasal cavity model were conducted. In this study, preliminary studies on the toxicity of the various formulation components (excipients and active ingredient) were carried out. Results from these studies indicated that for both the excipients and the drug, pH significantly affects the ciliary motility hence all ciliary beat frequency determinations were conducted at nasal pH. Furthermore, effects of the various concentrations (0.0625%(w/v), 0.125%(w/v), 0.25%(w/v), 0.5%(w/v) and l%(w/v)) of the excipients on ciliary motility were investigated. Transmission electron microscopy (TEM) studies proved useful in evaluating the integrity and changes in the surface morphology of the rat nasal mucosa post treatment with the various excipients (carboxymethyl cellulose, hydroxypropylmethyl cellulose, trimethyl chitosan 36.3% DQ, Carbopol P934 and polysorbate-80) at varying concentrations. Of the excipients investigated, hydroxypropylmethyl cellulose (HPMC) showed ciliofriendliness since there was no apparent ultra structural damage, although a slight decrease in ciliary beat frequency (CBF) was observed at the highest viscosity. Moreover, hydroxypropylmethyl cellulose (HPMC) is said to be a bioadhesive excipient, which would therefore confer its bioadhesive properties to the intranasal preparation to enhance the retention time between the absorbing mucosa and the drug and hence increase nasal drug absorption. This excipient was therefore selected as the ideal for use in the formulation of the intranasal preparation. The aqueous solubility of a drug plays an important role in nasal administration since it is required that the drug component be applied in a limited volume of about 200pl. To enhance the aqueous solubility of the sparingly water-soluble cyclizine HCl, a lactate salt was synthesised and characterised. This compound was found to be highly soluble in water. The intranasal preparation was therefore manufactured using the lactate form of cyclizine. A single blind study was conducted to determine and compare the pharmacokinetic parameters for both Valoid oral tablets containing 100mg cyclizine HCl (reference drug) and cyclizine lactate intranasal preparation 125mglml (study drug). The results obtained indicated a significant improvement in the bioavailability of cyclizine. For oral administration Cmax = 200.79ng/ml at tmax = 5.57h and for the intranasal preparation Cmax = 5354.22ng/ml at tmax = 1.59h. A 19.2-fold increase in drug bioavailability was observed after intranasal administration (AUCin = 122860.70ng/ml/h) compared with oral administration (AUCpo = 5943.48ng/ml/h). This enhanced bioavailability through nasal administration indicated that enhanced nasal drug absorption and hence increased bioavailability not only depends on the favourable anatomical and physiological characteristics of the nasal mucosa but possibly on the inherent physico-chemical characteristics of the drug molecule and the formulation components. Thus chemical modification of the sparingly water-soluble cyclizine HCl to the highly water-soluble cyclizine lactate facilitated the dissolution of more solute in a limited volume of solvent. This new feature therefore may have impacted positively to the transport of cyclizine across the nasal mucosa. Furthermore, the hydroxypropylmethyl cellulose (HPMC), component of the formulation, could have conferred its mucoadhesive properties to the preparation. Perhaps it increased the retention time of the dosage form within the nasal passages through bond formation with the nasal mucosa thereby increasing the contact time between the absorbing mucosa and the dosage form. This interaction between the mucoadhesive and the nasal mucosa may have resulted in the modification of tissue permeability (possibly transient opening of the tight junctions) and eventual increase in the drug penetration/absorption. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2004.

En Chile, Eucalyptus globulus es una de las especies forestales de mayor importancia, debido a su rápido crecimiento y a la calidad de su madera. Estas características hacen que sea una especie de alto rendimiento y de principal interés para la industria de la celulosa. Sin embargo, la especie resulta extremadamente sensible a las bajas temperaturas, por lo que ocurren grandes pérdidas en plantaciones jóvenes de Eucalyptus globulus. En este contexto, la biotecnología forestal se perfila como una herramienta eficaz para comprender y proponer soluciones a éste y a otros problemas. En el plano de la investigación científica, se han realizado importantes avances en la determinación de los mecanismos moleculares involucrados en la respuesta a estrés por frío en Eucalyptus globulus. Entre ellos, el aislamiento y secuenciación de una serie de genes involucrados en este proceso, como el gen EgCBF-1. Este gen codifica para un factor de transcripción que es activado por frío y que es capaz de activar numerosos genes de respuesta a estrés abiótico. En esta tesis se realizó un análisis estratégico que incluyó un recuento del panorama actual de la industria forestal chilena y los principales avances biotecnológicos atingentes. A través del análisis de la cadena de valor, se propuso un plan de negocios para la introducción de una variedad de Eucalyptus globulus tolerante a las bajas temperaturas en la industria forestal chilena. Se determinó que las principales ventajas para el desarrollo de este proyecto son las políticas gubernamentales favorables que incentivan el desarrollo biotecnológico del sector forestal y la consistencia de la industria forestal chilena. Se determinó que las principales desventajas son la carencia de una metodología para la transformación de especies de eucaliptos y la escasez de información a nivel social y legislación relativa al uso de organismos genéticamente modificados. La parte experimental de este trabajo de tesis se orientó al análisis funcional y estructural del promotor del gen EgCBF-1 en respuesta al estrés por frío. Se aisló y secuenció el promotor EgCBF-1 y se desarrollaron construcciones para estudios de funcionalidad. Se obtuvo una secuencia de 776 pares de bases río arriba del sitio de inicio de la transcripción del gen CBF-1 de Eucalyptus globulus, correspondiente a un segmento del promotor de dicho gen. En cuanto al análisis estructural, se confirmó la presencia de una serie de elementos de respuesta a estrés, entre ellos, cinco sitios de unión para el factor de transcripción EgICE-1, cuyo rol es clave en la respuesta al estrés provocado por las bajas temperaturas. Se determinó además, que éste y otros elementos de respuesta, están también presentes en promotores de genes ortólogos al promotor EgCBF-1. Se utilizaron dos estrategias para evaluar la funcionalidad del promotor: ensayos de funcionalidad mediante transformación transitoria en Nicotiana benthamiana y ensayos en plantas trnasgénicas de Arabidopsis thaliana. En ambos casos se utilizó el gen reportero GUS bajo el control del promotor en estudio.

Dans ces dernières années, les Systèmes de Transport Intelligents (STI) ont été considérés comme l'un des domaines de recherche les plus émergents en raison de leur rôle prometteur dans l'amélioration de la gestion du trafic et de la sécurité routière. Les applications coopératives de sécurité, étant les plus cruciales, ont gagné beaucoup d'intérêt. L'efficacité de ces applications dépend largement de l'échange efficace de deux principaux types d'informations. L'information de localisation périodique correspondant à l'information de localisation du voisinage et l'information événementielle qui est transmise en multi-sauts et générée lors de la détection d'une situation d'urgence. En raison de la caractéristique à grande échelle des STI, cette information fait l'objet du problème de congestion dans le réseau. L'objectif de cette thèse est d'assurer un contrôle fiable et robuste des informations de sécurité permettant de réduire la congestion du canal tout en tenant en compte des exigences des applications de sécurité. Nous examinons la diffusion de l'information événementielle en proposant une approche a multi-sauts qui a montré une amélioration de la réception de l'information. Cependant, cette approche reste très sensible à la charge de canal résultant de transmissions de l'information de localisation périodiques. D'autre part, la transmission efficace de l'information événementielle repose essentiellement sur la détection précise des événements de sécurité et en conséquence sur la précision de l'information de localisation. Ainsi, nous proposons un mécanisme de contrôle de l'information de localisation afin de fournir une meilleure précision et limiter la charge du canal. Les approches proposées dans cette thèse ont profondément étudié le compromis entre le respect des exigences des applications de sécurité et la gestion efficace de la congestion dans le réseau véhiculaire. / In the last decades, Intelligent Transportation Systems (ITS) have been considered as one of the most emerging research area due to their promising role in promoting traffic efficiency and enhancing road safety. ITS cooperative safety applications, being the most vital and critical, have gained a lot of attention. The effectiveness of these applications depends widely on the efficient exchange of two main types of information. The periodic awareness corresponding to the one-hop location information of surrounding environment and the multi-hop event-driven information generated at the detection of a safety situation. Due to the large scale characteristic of ITS, this information is expected to be subject to severe congestion which might impact its reliable reception. The goal of this thesis is to focus on the reliable and robust control of safety-related information by reducing the channel congestion and at the same time taking into account the requirements of safety applications. We address first the event-driven safety information. We proposed a multi-hop policy showed to improve the dissemination of the event-driven information. However, it remains strongly sensitive to the channel load resulting from periodic awareness transmissions. On the other hand, the effective transmission of event-driven information depends primarily on the accurate detection of safety events and accordingly on the accuracy of awareness. Thus, we provide an efficient awareness control mechanism in order to provide better accuracy and limit the channel congestion. The approaches proposed in this thesis have deeply investigated the trade-off between ensuring the requirements of cooperative safety and the efficient management of congestion in vehicular network.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19368号 / 医博第4045号 / 新制||医||1011(附属図書館) / 32382 / 新制||医||1011 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 吉村 長久, 教授 山田 泰広 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.